Epoxy-α-lapachone has in vitro and in vivo anti-leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm(2)), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm(2)) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm(2)). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

Efficacy of the treatment using a microemulsion loaded with epoxy-α-lapachone in combination with meglumine antimoniate against murine infection by Leishmania (Leishmania) amazonensis.

Leishmaniasis is a disease caused by Leishmania spp., affecting millions of people around the world. For decades, its treatment has been based on pentavalent antimonials, which notoriously cause toxic side effects in patients. In this study, epoxy-α-lapachone incorporated into an oil-in-water-type microemulsion (ELAP-ME) and meglumine antimoniate (MA) were assayed in monotherapy and in combination (ELAP-ME/MA) in BALB/c mice infected with Leishmania (Leishmania) amazonensis. In general, there was a reduction in paw lesion size (up to 37% reduction) and decreases of parasite loads in the footpad (∼40%) and lymph nodes (∼31%) of animals treated with ELAP-ME/MA, when compared to the non-treated control groups. Analyses of serum biochemical parameters revealed that the ELAP-ME/MA showed lower renal and hepatic toxicity when compared to MA 2-doses/week monotherapy. These findings indicate that the ELAP-ME/MA combination may be a promising approach for the treatment of cutaneous leishmaniasis.

Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition.

Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min-1 mg protein-1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min-1 mg protein-1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min-1 mg protein-1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.